Geldanamycin Enhances the Radioprotective Effect of Peroxyredoxin 6 in Irradiated 3T3 Fibroblasts.

The aim of the study was to evaluate the possibility of increasing the radioprotective potential of peroxiredoxin 6 (Prdx6) and its mutant form S32A by their combined use with geldanamycin (GA) for 3T3 fibroblasts irradiated with X-rays at a dose of 6 Gy. The mutant enzyme S32A, which does not have phospholipase activity, exhibits a more pronounced radioprotective activity when combined with GA. The use of this combination of radioprotective drugs completely abolishes the peak of NF-κB activity in irradiated 3T3 cells. Another transcription factor, p53, which is an indicator of the level of cell apoptosis and increases upon irradiation, is also reduced by S32A in combination with GA. The low-molecular-weight protein p21, which is a marker of cell senescence and whose production increases upon irradiation, is also normalized when S32A is used in combination with GA. In addition, the use of this combination of radioprotective drugs significantly reduces the stress response of 3T3 cells to X-ray irradiation.

Influence of Xenon on Survival of Sperm of Common Frog Rana temporaria during Slow Freezing.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

Participation of Hsp70 and Hsp90α Heat Shock Proteins in Stress Response in the Course of Type 1 Diabetes Mellitus.

The role of two heat shock proteins, Hsp70 and Hsp90α, on stress response in mice with severe diabetes mellitus induced by a high dose of alloxan (500 mg/kg body weight), as well as in RIN-m5F ß cells cultured in the presence of cytokines (IL-1 and TNF-α) was studied. Our results showed that severe type 1 diabetes mellitus (T1D) caused a higher expression of Hsp90α, but not Hsp70. Moreover, injections of the peroxiredoxin 6 antioxidant enzyme (PRDX6) did not affect the expression of these chaperones. Conversely, pro-inflammatory cytokines added to ß-cells caused a significant increase in the expression of Hsp90α and, substantially, Hsp70. Moreover, cells cultivated in the presence of PRDX6 were more susceptible to the cytokine effect. Thus, in the course of severe alloxan-induced T1D, no protective role of the heat shock proteins, was revealed, and their expression level was not increased by PRDX6. At the same time the protective potential of these proteins was shown in vitro with the use of RIN-m5F ß cells. Thus, the system of heat shock proteins was unable to prevent the devastating effects of severe T1D accompanied by high animal mortality.

Activation of Signal Pathways of Apoptosis under Conditions of Prolonged ER-Stress Caused by Exposure of Mouse Testicular Teratoma Cells to Selenium-Containing Compounds.

Aim to study the molecular mechanisms of apoptotic death of mouse testicular teratocarcinoma cells (line F-9) under exposure to the widely used selenium-containing compounds with antitumor activity, sodium selenite and methylseleninic acid. Methods fluorescence microscopy, MTT assay, Western blotting. Results It was shown that sodium selenite at a concentration of 10 µM and methylseleninic acid at concentrations of 1 and 10 µM cause apoptosis-dependent death of F-9 cells, excluding necrotic death. Western blotting showed an increase in the expression of XBP1s when treating F-9 cells with 1 µM methylseleninic acid. Conclusions 10 µM methylseleninic acid leads to cell apoptosis, most likely by activation of the IRE1 signaling pathway under prolonged stress of the endoplasmic reticulum.

Protein Partners of Selenoprotein SELM and the Role of Selenium Compounds in Regulation of Its Expression in Human Cancer Cells.

The search for potential partners of human SELM in lysates of two cancer cell lines, HT-1080 (fibrosarcoma) and MCF-7 (breast adenocarcinoma), was carried out. Two cytoplasmic actin isoforms-cytoplasmic actin 1 (cytoskeleton ß-actin) and cytoplasmic actin 2 (cytoskeletal γ-actin)-were identified as partners. In addition, the influence of two widely used antitumor selenium compounds (sodium selenite and methylseleninic acid) on the expression of SELM in cancer cells was studied. On the basis of the results obtained by real-time PCR and Western blotting, we concluded that 1 µM and 10 µM sodium selenite did not affect the expression of SELM in fibrosarcoma cells, whereas in breast adenocarcinoma cells 1 µM sodium selenite slightly increased expression and 10 µM sodium selenite significantly (approximately 2 times) decreased it. Methylseleninic acid in both cancer cell lines increased the SELM gene expression; the most pronounced effect was observed when fibrosarcoma cells were treated with 10 µM MSA (the expression of the hSelm gene increased almost 4 times).

Protective and adaptogenic role of peroxiredoxin 2 (Prx2) in neutralization of oxidative stress induced by ionizing radiation.

A radioprotective effect of exogenous recombinant peroxiredoxin 2 (Prx2) was revealed and characterized using an animal model of whole body X-ray irradiation at sublethal and lethal doses. Prx2 belongs to an evolutionarily ancient family of peroxidases that are involved in enzymatic degradation of a wide variety of organic and inorganic hydroperoxides. Apart from that, the oxidized form of Prx2 also exhibits chaperone activity, thereby preventing protein misfolding and aggregation under oxidative stress. Intravenous administration of Prx2 in animals at a concentration of 20 µg/g 15 min before exposure to ionizing radiation contributes to a significantly higher survival rate, suppresses the development of leucopenia and thrombocytopenia, as well as protects the bone marrow cells from genome DNA damage. Moreover, injection of Prx2 leads to suppression of apoptosis, stimulates cell proliferation and results in a more rapid recovery of the cell redox state. Exogenous Prx2 neutralizes the effect of the priming dose on the second irradiation of the cells. The radioprotective properties of exogenous Prx2 are stipulated by its broad substrate peroxidase activity, chaperone activity in the oxidized state, and are also due to the signal-regulatory function of Prx2 mediated by the regulation of the level of hydroperoxides as well as via interaction with redox-sensitive regulatory proteins.

Influence of Sodium Selenite on the mRNA Expression of the Mammalian Selenocysteine-Containing Protein Genes in Testicle and Prostate Cancer Cells.

The sodium selenite concentration that reduces the viability of Du-145 human prostate adenocarcinoma cells and F-9 mouse testicular teratocarcinoma cells was determined. We investigated the effect of sodium selenite on the mRNA expression level of the genes encoding mammalian selenocysteine-containing glutathione peroxidases and thioredoxin reductases (key antioxidant enzymes involved in the regulation of intracellular thiol redox balance), endoplasmic reticulum selenoproteins, and selenoproteins located in the testes and prostate.

Involvement of the p38 MAPK signaling cascade in stress response of RAW 264.7 macrophages.

The role of the p38 MAPK signaling cascade was studied in stress response of RAW 264.7 macrophages to extremely low-intensity centimeter microwaves. Irradiation stimulated production of a number of cytokines (IL-1, IL-6, TNF-α, INF-γ and IL-10), as well as induced activation of the signaling cascades NF- κB and p38 MAPK, and enhanced expression of Hsp72 heat shock protein. In the presence of the cascade p38 MAPK inhibitor (p38 MAP kinase inhibitor XI), the stimulating effects of electromagnetic waves were abrogated either completely (for NF-κB and Hsp72) or partially (for p38 MAPK and cytokines). The results obtained are indicative of a high sensitivity of the signaling cascade p38 MAPK to the effect of low-intensity physical fields.

Cloning, intracellular localization, and expression of the mammalian selenocysteine-containing protein SELENOI (SelI) in tumor cell lines.

The intracellular localization of human selenoprotein SelI and the degree of expression of its gene in different human tumor cell lines were determined. It was found that the SelI protein is present in the nucleus, cytoplasm, and endoplasmic reticulum and is absent in the nucleolus. Since the oxidative stress caused by a sharp increase in the content of free radicals in the body is one of the causes of malignant transformation, the study of the role of the trace element selenium and selenocysteine-containing proteins as antioxidants in carcinogenesis is of great scientific interest.

Seasonal changes in proteolytic activity of calpains in striated muscles of long-tailed ground squirrel Spermophilus undulatus.

Seasonal changes in proteolytic activity and content of calpains in striated muscles of the longtailed ground squirrel Spermophilus undulatus were studied by casein zymography and Western blotting analysis. The results testify to hyperactivation of calpain proteases in the skeletal muscles of awakened animals during the "winter" activity. The observed changes are discussed in the context of adaptation of skeletal muscles of long-tailed ground squirrels to hibernation.

The role of peroxiredoxin 6 in neutralization of X-ray mediated oxidative stress: effects on gene expression, preservation of radiosensitive tissues and postradiation survival of animals.

Peroxiredoxins are redox-sensing multifunctional enzymes, among them peroxiredoxin 6 (Prx6) can neutralize the most broadest range of hydroperoxides and play an important role in maintaining the redox homeostasis of the cell. In the present study, radioprotective and signaling regulatory effects of Prx6 were demonstrated and characterized. Intravenously administered exogenous Prx6 protects the organism of mice from the destructive action of ionizing radiation in the lethal dose range of 5-10 Gy. Dose reduction factor of 1.4 Prx6 injection reduces the severity of radiation-induced leuko- and thrombopenia in irradiated animals, also preventing the destruction of epithelial cells in the small intestine. Injecting exogenous Prx6 also as its mutated form of Prx6-C47S lacking peroxidase activity affects the expression of genes involved in antioxidant response, DNA reparation, apoptosis and inflammatory processes, in bone marrow cells both in intact animals and in those subjected to ionizing radiation. The radioprotective properties of Prx6 are based, on the one hand, on the capability for ROS neutralization, and on the other hand - on the potentiality for activation of reparation processes of the cell under oxidative stress conditions. Prx6 can be considered as a potentially perspective radioprotective agent for the reduction of risks from the damaging action of ionizing radiation on the mammalian organism.

Fatty acids and cholesterol in the liver cell nuclei of hibernating Yakutian ground squirrels.

The content of neutral lipids in tissue homogenates and liver cell nuclei of hibernating Yakutian ground squirrels was studied. In homogenates, hibernation increases the content of fatty acids and reduces the content of glycerides and cholesterol. When studying the liver cell nuclei of torpid winter ground squirrels, we detected a twofold increase in the content of fatty acids, cholesterol, and monoglycerides as compared to the "summer" ground squirrels. In the active "winter" ground squirrels, as compared to the torpid winter ones, the content of cholesterol did not change, whereas the content of fatty acids, monoglycerides, and diglycerides decreased but remained higher than in the "summer" ground squirrels.

Droplet-like heterogeneity of aqueous tetrahydrofuran solutions at the submicrometer scale.

A droplet formation in aqueous solutions of tetrahydrofuran (THF) has been experimentally detected at the submicrometer scale using two independent laser diagnostic techniques (dynamic light scattering and laser phase microscopy) and described in terms of THF-water intermolecular hydrogen bonding. It is shown that the nanodroplets have a mean size of 300 nm, their refractive index is higher than that of the ambient liquid, and they are highly enriched with THF molecules. The maximum of light scattering intensity falls within the THF concentration range 2-8 mol. %, which corresponds to the volume number density of the nanodroplets ∼1010-1011 cm-3. A theoretical explanation of forming the nanodroplets with a high content of THF, which is based on a model of dichotomous noise being applied to the so-termed "twinkling" hydrogen bonds and involves spinodal decomposition in the unstable region enclosed within the dichotomous binodal, is proposed. The parameters of hydrogen bonds in the molecular system "water-THF" were found, and the phase diagram of the solution with allowance for cross-linking hydrogen bonds was constructed.

Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel.

In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active "winter" ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the "summer" ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.

Expression of human selenoprotein genes selh, selk, selm, sels, selv, and gpx-6 in various tumor cell lines.

The expression level of the genes encoding six selenocysteine-containing human proteins was determined in the brain, cervical, liver, breast, prostate, and human fibrosarcoma cancer cells. It was found that a high level of expression in all studied types genes of tumor cells is characteristic for selh, selk, and selm genes, encoding SelH, SelK, and SelM proteins, respectively, whereas a complete lack of such expression was shown for gpx-6, selv, and sels genes. The results of this work can be regarded as a major prerequisite for further studies on the role of the three selenoproteins SelH, SelK, and SelM in the regulation of carcinogenesis processes associated with these types of cancer.

Peroxiredoxin 6 is a natural radioprotector.

After injection of 20 mg/kg peroxiredoxin 6 to male Kv:SHK mice 15 min before X-ray irradiation in the range of lethal doses (7-10 Gy), the mice remained alive for 30 days, whereas the mortality of the control animals was 100%. In the irradiated animals, peroxiredoxin 6 decreased the severity of radiation-induced leucopenia, granulocytopenia, and thrombocytopenia, increased the number of blood corpuscles, and prevented the mass death of epithelial cells and the destruction of the small intestine. Thus, peroxiredoxin 6 can be regarded as a prophylactic radioprotective agent.

[Selective Heating of Membrane-forming Holes in Teflon Film Exposed to Decimeter Waves].

Calculations of heating of membrane-forming holes in Teflon film exposed to decimeter waves were performed. The dependence of the temperature increment in holes on the geometry of holes, electrolyte concentration, and decimeter wave frequency was studied. The kinetics of heating depending on the hole diameter was also obtained. It was concluded that the observed in the experiment effects of the decimeter wave on bilayer lipid membranes resulted from the elevated concentration of decimeter electromagnetic waves in membrane-forming hole that led to selective heating of electrolyte in hole and bilayer lipid membranes.

[Prospects for Application of Gases and Gas Hydrates to Cryopreservation].

In the present review, we tried to evaluate the known properties of gas hydrates and gases participating in the formation of gas hydrates from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the papers on freezing biological materials in the presence of inert gases, and to analyze the perspectives for the development of this direction. For the purpose, we searched for the information on the physical properties of gases and gas hydrates, compared processes occured during the formation of gas hydrates and water ice, analyzed the influence of the formation and growth of gas hydrates on the structure of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide especially on hypothermal conditions and probable application of these properties in cryopreservation technologies. The description of the existing experiments on cryopreservation of biological objects with the use of gases was analyzed. On the basis of the information we found, the most perspective directions of work in the field of cryopreservation of biological objects with the use of gases were outlined. An attempt was made to forecast the potential problems in this field.

[On the Effect of α-Tocopherol on Protein Kinase C Activity in vitro].

The effect of the antioxidant α-tocopherol on rat brain protein kinase C activity as a model to study bimodal dose-dependent effect has been investigated. Enzyme activity has been monitored photometrically with a luciferase reporter assay that measures ADP produced by posphorylation. The inhibition of protein kinase C activity by α-tocopherol was found at the concentration range from 10(-3) to 10(-6) M with no effect of ultra low doses of the antioxidant (below. 10(-12) M). The absence of bimodal dose-dependent effect may be associated with the enzyme source.

The role of CK2 protein kinase in stress response of RAW 264.7 macrophages.

The role of casein kinase 2 (CK2) in the activation of the key NF-κB signaling cascade and other signaling and stress proteins during stress induced by exposure to nonthermal low-intensity electromagnetic radiation has been investigated. The ability of CK2 to regulate the activation of the NF-κB signaling cascade and upregulate the proinflammatory cytokine production and TLR4 receptor expression has been demonstrated. The existence of an alternative signaling pathway for NF-κB cascade activation directly involving protein kinase CK2 has been demonstrated using inhibition analysis in cells stressed by microwave irradiation.